Bone substitute compositions, methods of preparation and clinical applications

ABSTRACT

The present invention relates to bone substitute compositions and methods of their preparation, and their use in a wide variety of clinical applications. The compositions include calcium phosphate, acidic calcium salt, basic calcium salt, sodium hydrogen phosphate and porogen. The compositions further include a mixing liquid. The compositions can optionally include biological signaling molecules and/or a growth compound. Further, the compositions can optionally include a plasticizer.

This patent application claims the benefit of U.S. patent applicationSer. No 13/711,261 filed on Dec. 11, 2012, entitled “Bone SubstituteCompositions, Methods of Preparation and Clinical Applications” which iscurrently pending, U.S. patent application Ser. No. 12/882,554 filed onSep. 15, 2010, entitled “Bone Substitute Compositions, Methods ofPreparation and Clinical Applications” which issued as U.S. Pat. No.8,357,364 on Jan. 22, 2013, and U.S. Provisional Patent Application Ser.No. 61/242,596 filed on Sep. 15, 2009, entitled “NanostructuredInjectable Bone Cement for Bone Regeneration”.

GOVERNMENT CONTRACT

The research conducted for this invention was partially funded undergrants (Nos. DAMD 17-02-1-0717, SAP-4100041556 and SAP-410045998) and acontract (No. DAMD 17-02-0717) from the government of the United Statesand therefore, the government of the United States has certain rights inthis invention.

FIELD OF THE INVENTION

The present invention relates to bone substitute compositions, methodsfor their preparation, and their use in clinical applications. Thecompositions are particularly suitable for use in bone regeneration,bone tissue engineering and as a delivery vehicle for cellular andbiological materials.

BACKGROUND OF THE INVENTION

Various polymer- and ceramic-based scaffolds have been studied for useas bone substitute compositions and for the purpose of boneregeneration. These scaffolds include hydroxyapatite (HA)-based ceramicsand calcium phosphate cements (CPCs). CPCs are generally moldable,putty-like compounds that can be easily introduced into a defective bonesite. They typically set within a period of time at normal bodytemperatures to form mechanically stable, osteo-conductive orosteo-inductive materials. Additionally, CPCs convert into naturalbone-like calcium-deficient hydroxyapatite (CDHA) in vivo making themattractive for bone tissue engineering. Currently used CPCs have arelatively long setting time, for example, in excess of several tens ofminutes, slow conversion, low resorption rates and low bone regenerationrates. CPCs would be more attractive for clinical applications if theyexhibited enhanced resorption-degradation characteristics in vivo.

Further, the polymer and ceramic cement-based scaffolds that are knownin the art for use as bone substitute compositions are generally notamenable for in situ incorporation of cells, growth factors and/orbiological systems. This may be due, at least in part, to the harshreaction conditions or reagents that are used and may be toxic to cellsand biological systems. Hence, as a result, the cellular or biologicalcomponents could be incorporated into the pre-fabricated system whichwould not allow one to substantially control the amount, distributionand homogeneity of the delivery agents. Further, the acidic or basicdegradation products could also prove to be harmful to the cells orbiological molecules added to the pre-fabricated system.

Reconstructive surgery in recent years has focused intensely on tissuerepair and regeneration, in an attempt to overcome the limitations ofthe current treatment strategies. Artificial tissue substitutes havesignificantly assisted surgeons in the restoration of the form and,partly, the function of defective bones. In this context, theimplementation of bioresorbable scaffolds have been regarded as anoptimal model for tissue regeneration.

There is a need in the art to design and develop bone substitutecompositions which can be generated under physiological conditions ofneutral pH and may demonstrate the ability to contain nano-carriers ofcalcium phosphate that are complexed to cellular and/or biologicalmaterials, such as DNA, growth factors, cells and proteins.Additionally, it is desired for bone substitute compositions to have areasonably short setting time, a reasonably rapid conversion time andrelatively high resorption and bone regeneration rates.

Further, it is desired for bone substitute compositions to haverelatively short initial setting times and relatively short finalsetting times; improved porosity of the CPCs, i.e. increased number andsize of the pores inside the cement to accelerate bone tissueinfiltration; increased exposed area to lead to greater resorptionrates; and the ability to directly incorporate various cellular and/orbiological materials in the developed apatitic—CPC to induce rapid boneregeneration around a defective bone site. Furthermore, it is desiredthat the incorporation of porosity, various cellular and/or biologicalmaterials including carriers of DNA, growth factors, proteins, anddrugs, not impact the setting characteristics and mechanical propertiesof the bone substitute composition.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a bone substitutecomposition including a powder component and a liquid component. Thepowder component includes calcium phosphate, acidic calcium salt, basiccalcium salt, material selected from monosodium hydrogen phosphate,disodium hydrogen phosphate and mixtures thereof, and porogen. Theliquid component includes a colloidal mixture including nanoparticulatecalcium phosphate and calcium salt. The bone substitute composition iseffective to regenerate bone in the absence of a biological growthcomponent.

The bone substitute composition can further include a plasticizer. Theplasticizer can be present in the powder component of the bonesubstitute composition.

The calcium phosphate can be selected from the group of monocalciummonophosphate anhydrite, calcium hydrogenphosphate dihydrate, calciumhydrogenphosphate anhydrite, hydroxyapatite, alpha-tricalcium phosphate,beta-tricalcium phosphate, fluorapatite, octacalcium phosphate,tetracalcium phosphate, carbonated calcium phosphate, and mixturesthereof. The calcium phosphate can include alpha-tricalcium phosphate.

The acidic calcium salt is selected from the group of calcium sulfate,calcium hydrogenphosphate dihydrate, calcium hydrogenphosphateanhydrite, calcium oxalate, calcium citrate, calcium tartrate, calciumpicrate and mixtures thereof.

The basic calcium salt is selected from the group of calcium carbonate,calcium bicarbonate, calcium dihydroxide, and mixtures thereof.

The porogen is selected from the group of recrystallized organic salt,recrystallized inorganic salt, engineered peptide, natural polymer, acomposite of natural and synthetic polymers, synthetic biodegradablepolymer, natural extra cellular matrix protein, and mixtures thereof.

The absence of the biological growth component in the composition caninclude the absence of BMP-2.

The composition can have a porosity of about 80 percent or greater.

In another aspect, the present invention provides a bone substitutecomposition including a powder component and a liquid component. Thepowder component includes calcium phosphate, acidic calcium salt, basiccalcium salt, material selected from monosodium hydrogen phosphate,disodium hydrogen phosphate, and mixtures thereof, and porogen. Theliquid component includes a colloidal mixture including nanoparticulatecalcium phosphate and calcium salt. The porogen is present in an amountsuch that the composition has a porosity of about 80% or greater.

The composition can have a surface area of from about 60 m²/gram toabout 120 m²/gm.

The composition can further comprise a plasticizer.

The composition can be osteo-inductive.

In yet another aspect, the present invention provides a method ofpreparing a bone substitute material. The method includes combiningcalcium phosphate, acidic calcium salt, basic calcium salt, materialselected from monosodium hydrogen phosphate, disodium hydrogen phosphateand mixtures thereof, and porogen to form a powder component, and mixingthe powder component with a liquid colloidal mixture includingnanoparticulate calcium phosphate and calcium salt, the colloidalmixture being complexed with at least one of a compound selected fromthe group consisting of protein, peptide, DNA, drug, stem cell, normalcell, and combinations thereof. The bone substitute material iseffective to regenerate bone in the absence of a biological growthcomponent.

The bone substitute compositions described above can be used in clinicalapplications to repair or replace bone material.

BRIEF DESCRIPTION OF THE FIGURES

The invention as set forth in the claims will become more apparent fromthe following detailed description of certain preferred practicesthereof illustrated, by way of example only, and the accompanyingfigures, wherein:

FIG. 1 is a plot showing the change of pH with time for various bonesubstitute compositions.

FIG. 2 is a plot showing x-ray diffraction patterns at various agingtimes for a bone substitute composition in accordance with an embodimentof the present invention.

FIGS. 3( a), 3(b) and 3(c) are SEM images at various aging times for abone substitute composition in accordance with an embodiment of thepresent invention.

FIGS. 4( a) and 4(b) are plots showing x-ray diffraction patterns atvarious aging times for bone substitute compositions in accordance withembodiments of the present invention.

FIGS. 5( a), 5(b) and 5(c) are SEM images at various aging times for abone substitute composition in accordance with an embodiment of thepresent invention.

FIGS. 6( a), 6(b), 6(c) and 6(d) are SEM images at various aging timesfor a bone substitute composition in accordance with an embodiment ofthe present invention.

FIG. 7( a) is a bar graph showing cell proliferation on various bonesubstitute compositions including bone substitute compositions inaccordance with embodiments of the present invention. FIG. 7( b) is aplot showing a release profile of BSA from a bone substitute compositionin accordance with an embodiment of the present invention.

FIGS. 8( a) and (b) are digital images showing bone substitutecompositions in accordance with embodiments of the present invention.

FIG. 9 is a digital image showing printed scaffolds of a bone substitutecomposition in accordance with an embodiment of the invention.

FIGS. 10( a) and 10(b) are micro-CT images of an implanted bonesubstitute composition in accordance with an embodiment of the presentinvention, FIG. 10( c) is a histological assessment showing new boneformation for a bone substitute composition in accordance with anembodiment of the present invention.

FIG. 11( a) is a histological assessment showing new bone formation fora bone substitute composition in accordance with an embodiment of thepresent invention. FIG. 11( b) is a micro-CT and picture showing anexplant and implant of a bone substitute compositions in accordance withan embodiment of the present invention. FIG. 11( c) is a radiography ofan implanted bone substitute composition in accordance with anembodiment of the present invention.

FIG. 12 is a x-ray radiograph showing an implant and a picture showingthe retrieved implant of a bone substitute composition in accordancewith an embodiment of the present invention.

FIG. 13 is a micro-CT assessment (photographs) showing a comparison of aknown composition with bone substitute compositions in accordance withembodiments of the present invention.

FIG. 14 is a histological assessment for an implant of a bone substitutecomposition in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides bone substitute compositions, methods fortheir preparation and their use in various clinical applications. Thebone substitute compositions of the present invention are suitable foruse in clinical applications to repair or replace defective native bonetissue. The detective native bone tissue can be in orthopedic, dentaland craniofacial areas of a patient. The bone substitute compositionsare effective to regenerate bone. Thus, the bone substitute compositionsof the present invention are osteo-inductive. The bone substitutecompositions are particularly suitable for use as a scaffold, forexample, in a defective bone site. The bone substitute compositions canbe in various forms, such as, but not limited to, paste, putty andcement. For example, the bone substitute compositions can be prepared inthe form of a paste or putty and upon setting can form a cement. Thephysical characteristics of each of the paste, putty and cement canvary. The bone substitute compositions can be placed in vivo in apatient using various techniques known in the art, such as, by injectingor implanting the bone substitute composition into the patient. Further,the bone substitute compositions of the present invention maydemonstrate advantageous characteristics, such as, but not limited to,excellent cell and host tissue biocompatibility. Furthermore, the bonesubstitute compositions of the present invention may demonstrate theabove-mentioned advantageous characteristics in the absence of abiological growth component.

The bone substitute composition of the present invention includes apowder component and a liquid component. The powder component includescalcium phosphate, acidic calcium salt, basic calcium salt, materialselected from monosodium hydrogen phosphate, disodium hydrogen phosphateor a mixture thereof, and porogen. The liquid component includes amixing liquid.

The calcium phosphate can be selected from a variety of materials knownin the art and includes, but is not limited to, monocalciummonophosphate anhydrite, calcium hydrogen phosphate dihydrate, calciumhydrogen phosphate anhydrite, hydroxyapatite, alpha-tricalciumphosphate, beta-tricalcium phosphate, fluorapatite, octacalciumphosphate, tetracalcium phosphate, carbonate calcium phosphate, andmixtures thereof. In one embodiment, the calcium phosphate includesalpha-tricalcium phosphate. The calcium phosphate can be present in thepowder component of the bone substitute composition in varying amounts.It is contemplated that the amount of calcium phosphate employed maydepend on the specific starting compounds selected for use in the powdercomponent of the bone substitute composition of the present invention.In alternate embodiments, the calcium phosphate can be present in anamount of at least about 50% or no greater than about 98% by weightbased on total weight of the powder component of the composition. Infurther embodiments, the calcium phosphate can be present in an amountsuch that it constitutes from about 50% to about 98%, or from about 60%to about 95%, or from about 65% to about 90% by weight based on totalweight of the calcium phosphate, acidic calcium salt and basic calciumsalt.

The acidic calcium salt can be selected from a variety of materialsknown in the art and includes, but is not limited to, calcium sulfate,calcium hydrogen phosphate dihydrate, calcium hydrogen phosphateanhydrite, calcium oxalate, calcium citrate, calcium tartrate, calciumpicrate, and mixtures thereof. In one embodiment, the acidic calciumsalt includes calcium sulfate. The acidic calcium salt can be present inthe powder component of the bone substitute composition in varyingamounts. It is contemplated that the amount of acidic calcium saltemployed may depend on the specific starting compounds selected for usein the powder component of the bone substitute composition of thepresent invention. In alternate embodiments, the acidic calcium salt canbe present in an amount of at least about 10% or no greater than about20% or from about 10% to about 20%, by weight based on total weight ofthe calcium phosphate, acidic calcium salt and basic calcium salt.

The basic calcium salt can be selected from a variety of materials knownin the art and includes, but is not limited to, calcium carbonate,calcium bicarbonate, calcium dihydroxide, and mixtures thereof. In oneembodiment, the basic calcium salt includes calcium carbonate. The basiccalcium salt can be present in the powder component of the bonesubstitute composition in varying amounts. It is contemplated that theamount of basic calcium salt employed may depend on the specificstarting compounds selected for use in the powder component of the bonesubstitute composition of the present invention. In alternateembodiments, the basic calcium salt can be present in an amount of atleast about 5% or no greater than about 20% or from about 5% to about20%, by weight based on total weight of the calcium phosphate, acidiccalcium salt and basic calcium salt.

The porogen can be selected from a variety of materials known in the artand includes, but is not limited to, recrystallized organic salt,recrystallized inorganic salt, engineered peptide, natural polymer, acomposite of natural and synthetic polymers, synthetic biodegradablepolymer, natural extra cellular matrix protein, and mixtures thereof. Inone embodiment, the porogen is an inorganic salt, such as, but notlimited to, potassium chloride, sodium chloride, sodium phosphate,sodium citrate, sodium tartrate, sodium acetate. In one embodiment, theporogen is a natural polymer, such as, but not limited to, mannitol,collagen, sucrose, fibrin, gelatin, alginate, chitosan, fibrin-gelatincomposite, paraffin, polyol, poly lactic-co-glycolic acid (PLGA), andmixtures thereof. In another embodiment, the porogen is a syntheticbiodegradable polymer, such as, but not limited to, poly-lactic acid,poly-e-caprolactone, poly-lactic-co-glycolic acid, and mixtures thereof.In yet another embodiment, the porogen is a natural extra cellularmatrix protein, such as, but not limited to, urinary bladder matrix. Theporogen can be present in the powder component of the bone substitutecomposition in varying amounts. It is contemplated that the amount ofporogen employed may depend on the specific starting compounds selectedfor use in the powder component of the bone substitute composition ofthe present invention. In alternate embodiments, the porogen can bepresent in an amount of at least about 1% or no greater than about 60%by weight based on total weight of the total percentage of the calciumphosphate, acidic calcium salt and basic calcium salt, and the porogen.In further embodiments, the porogen can be present in an amount suchthat it constitutes from about 1% to about 60% or from about 10% toabout 60% or from about 30% to about 50%, by weight based on totalweight of the total percentage of the calcium phosphate, acidic calciumsalt and basic calcium salt, and the porogen. In yet another embodiment,the porogen is present in an amount such that the bone substitutecomposition of the present invention has a porosity of about 80 percentor greater. Without intending to be bound by any particular theory, itis believed that rendering the bone substitute composition porous in therange of about 80 percent or greater, enables cells to penetrate thepores and uptake nanoparticulate carriers (which will be later describedin detail) to produce an osteo-inductive composition.

The powder component of the bone substitute composition also includesmonosodium hydrogen phosphate, disodium hydrogen phosphate, or mixturesthereof. The amount can vary and in one embodiment can be present suchthat the monosodium hydrogen phosphate, disodium hydrogen phosphate, ormixtures thereof, constitutes from about 5% to about 20% by weight basedon total weight of the powder component.

In one embodiment, the powder component can optionally include calciumsalt. Suitable examples can include those known in the art. Further, theamount of calcium salt can vary depending on the volume of the liquidcomponent.

The mixing liquid in the liquid component of the bone substitutecomposition of the present invention includes a colloidal mixtureincluding nanoparticulate calcium phosphate and calcium salt. The amountof nanoparticulate calcium phosphate present in the colloidal mixturecan vary. Further, the amount of calcium salt in the colloidal mixturecan also vary. The colloidal mixture including nanoparticulate calciumphosphate and calcium salt can be in the form of a solution. Thesolution can be prepared using a variety of known methods. In alternateembodiments, preparation of the solution can be in accordance with theprocedure described in U.S. Pat. No. 7,247,288 and pending U.S.application Ser. No. 11/811,992 having Publication No. 2008/0095820 A1,which are both incorporated in their entirety by reference herein.

Furthermore, the pH of the colloidal mixture can vary. In alternateembodiments, the colloidal mixture can have a pH of from about 6.5 toabout 9 or from about 7.2 to about 7.4. In one embodiment, the colloidalmixture is complexed to at least one cellular and/or biological compoundwhich can include, but is not limited to, protein, peptide, DNA, drug,stem cell, normal cell and combinations thereof. The nanoparticulatecalcium phosphate in the colloidal mixture can serve as carriers ordelivery agents of these cellular and/or biological compounds.

Furthermore, the nanoparticulate calcium phosphate with the cellularand/or biological compounds complexed thereto can infiltrate the openpores of the bone substitute composition in vivo.

In one embodiment of the present invention, the bone substitutecomposition does not include a biological growth component, such as, butnot limited to, biological signaling molecules and/or a growth compound.Thus, the bone substitute composition is prepared, formed into a bonesubstitute cement and employed in a clinical application, in the absenceof a biological growth component, such as, but not limited to, BMP-2.

In an alternate embodiment of the present invention, the bone substitutecomposition includes a biological growth component, such as, but notlimited to, biological signaling molecules and/or a growth compound. Thebiological growth component can be selected from a variety of biologicalgrowth components known in the art. A suitable biological growthcomponent for use in the present invention includes, but is not limitedto, BMP-2. The biological growth component can be in the form of apowder and can be included in the powder component of the bonesubstitute composition.

In one embodiment, calcium sulfate and calcium carbonate are present inthe bone substitute composition in approximately equal amounts byweight. In a further embodiment, the calcium phosphate in the bonesubstitute composition constitutes at least about 50% by weight and thecalcium sulfate and calcium carbonate are in approximately equal amountsby weight.

In still another embodiment, the bone substitute composition includesfrom about 75% to about 85% by weight of calcium phosphate, from about8% to about 15% by weight of calcium sulfate, from about 8% to about 15%by weight of calcium carbonate, from about 5% to about 20% by weight ofmono- or di-sodium hydrogen phosphate or a mixture thereof, based ontotal weight of these compounds, and the total composition (thesecompounds and porogen) being from about 30% to about 50% by weightporogen.

In one embodiment, wherein the powder component of the bone substitutecomposition includes calcium phosphate, acidic calcium salt, basiccalcium salt, disodium hydrogen phosphate, and porogen, and the mixingliquid includes the use of a colloidal mixture including nanoparticulatecalcium phosphate and calcium salt solution, the amount of the disodiumhydrogen phosphate added to the powder component depends on the volumeand composition of the colloidal mixture including nanoparticulatecalcium phosphate and calcium salt solution in the liquid component. Thestoichiometric amount of disodium hydrogen phosphate required to convertthe excess Ca²⁺ into hydroxyapatite can be calculated in accordance withthe following equation:(M _(Ca))×10×Ca²⁺+(M_(PO))×6×HPO₄ ²⁻+H₂O→Ca₁₀(PO₄)₆(OH)₂

wherein M_(Ca) is the molarity of the calcium ion (i.e. concentration ofCa ions in moles/titer in the colloidal mixture of nanoparticulatecalcium phosphate solution) and V_(Ca) is the volume of the colloidalmixture of nanoparticulate calcium phosphate solution used. M_(PO) isthe concentration of phosphate ions in moles/liter required to convertthe certain volume and concentration of Ca-ions into hydroxyapatite. Thevalue of M_(PO) in grams is calculated in accordance with the followingequation:(M _(Ca))×10=(M_(PO))×6

In addition to this amount (M_(PO) in grams) of Na₂HPO₄, an excess (atleast 0.25 molar) of Na₂HPO₄ is used to catalyze the bone substitutecomposition reaction. Thus, the minimum amount of Na₂HPO₄ required toadd to the above mentioned bone substitute composition is (M_(PO)+0.25)moles for each liter of colloidal mixture of nanoparticulate calciumphosphate solution. Therefore, depending on the volume of colloidalmixture used for a specific bone substitute composition with a specificpowder to liquid ratio, the amount of Na₂HPO₄ can be calculated. Forexample, a colloidal mixture of nanoparticulate calcium phosphatesolution containing 0.2M CaCl₂ and colloidal nano-particles ofhydroxyapatite to be used as a liquid in the above mentioned powdercomposition with a powder to liquid ratio of between 2.33 g/cc and 1.51g/cc, requires addition of between 10% and 20% Na₂HPO₄, respectively (byweight of the powder composition weight, i.e. powder composition toNa₂PO₄ weight ratio is 10 to 5).

The powder component and the liquid component can be combined by simplemixing to form the bone substitute composition. Various known mixingtechniques, methods and equipment can be used. In one embodiment, thecomponents are combined at room temperature. Further, the order in whichthe various compounds in the powder component and the mixing liquid arecombined with each other is not critical. The combination of the powderand liquid components will result in product having various forms, suchas, for example, a paste or putty. The form of the product, and itsphysical characteristics, may depend on the amount of the powdercomponent and the amount of the liquid component used. For example, ahigher powder-to-liquid ratio can result in a dry paste and a lowerpowder-to-liquid ratio can result in a wet or liquid paste. The paste orputty product is allowed to set for a period of time and upon setting,the resultant product is in the form of a cement. Without intending tobe bound by any particular theory, it is believed that the presence ofcalcium salt, e.g., in the powder component or the liquid component orboth, assists in controlling the setting time of the resulting product.The cement product can then be implanted in vivo into a patient for useas a scaffold at a bone defect site.

In one embodiment, the ratio of powder component to liquid component isbetween about 1.50 g/ml:1.0 g/ml and 3.33 g/ml:1.0 g/ml. In thisembodiment, the initial setting time is below about ten minutes or aboutseven minutes and the final setting time is below about 30 minutes orabout 18 minutes. A higher ratio of powder component to liquid component(i.e., greater than 3.33 g/ml:1.0 g/ml) can result in a very dry pastehaving a setting time from about two to three minutes and therefore, maybe difficult to handle for clinical applications. A lower ratio ofpowder component to liquid component (i.e., less than 1.50 g/ml:1.0g/ml) can result in a very fluid paste which requires a significantlylonger setting time.

In one embodiment, the resulting bone substitute composition is amoldable, putty-like structure that sets within minutes. In a furtherembodiment, the composition sets within less than ten minutes. Inanother embodiment, the setting time is from five minutes to twelveminutes.

In still another embodiment, the bone substitute composition canoptionally include a plasticizer, e.g., a cohesion promoter. Withoutintending to be bound by any particular theory, it is believed that thepresence of the plasticizer assists the particles to coalesce togetherto enable the coalesced particles to flow plastically. The plasticizeralters the surface chemistry of the particles and does not affect thesetting characteristics of the bone substitute composition to form acement. The plasticizer can be selected from a variety of materialsknown in the art and includes, but is not limited to, sodium dextransulfate, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin,alginic acid, sodium salt, polyvinyl pyrrolidone, hyaluronic acid,potassium salt, chondroitin sulfate, chitosan lactate, hydroxypropylmethylcellulose, carboxymethyl cellulose and mixtures thereof. Theplasticizer can be present in the powder component or in the liquidcomponent or both. In a preferred embodiment, the plasticizer is presentin the powder component. The plasticizer can be present in variousamounts. It is contemplated that the amount of plasticizer employed maydepend on the specific starting compounds selected for use in the bonesubstitute composition. In a further embodiment, the plasticizer can bepresent in an amount of from about 2% to about 10% by weight based ontotal weight of the powder component without the weight of porogen.

The presence of plasticizer can result in the bone substitutecomposition having a viscosity which renders it injectable into a bone,e,g., bone defect site, of a patient from about one to five minutesafter initiating the mixing procedure of the powder and liquidcomponents. After injection, the injectable composition sets and forms acement in vivo, e.g., at the bone defect site within the patient.

In one embodiment, the resulting bone substitute composition, e.g.,cement, of the present invention has an increased surface area ascompared to the surface area of the starting powder component. Forexample, in one embodiment, the surface area of the resulting bonesubstitute cement is about sixty times the surface area of the startingpowder component. In a further embodiment, the surface area of theresulting bone substitute cement is about 60 m²/g and the surface areaof the starting powder component is about 1 m²/g or 2 m²/g. The surfacearea can be further enhanced by subjecting the precursors to mechanicalactivation. In one embodiment, when mechanical activation is employed,the surface area of the starting powder component can increase to about4 m²/g and the surface area of the resulting cement can increase to arange of about 90 to about 120 m²/g. Thus, the bone substitutecomposition can have a surface area of from about 60 m²/g to about 120m²/g.

The bone substitute composition of the present invention can be used inbone engineering and bone regeneration applications. The bone substitutecomposition can be applied to a defective bone site using varioustechniques, methods and equipment known in the art. In alternateembodiments, the bone substitute composition can be implanted into apatient at the defective bone site using conventional surgicaltechniques known in the art, or the bone substitute composition can beinjected into the defective bone site of the patient using conventionaltechniques including, but not limited to, a needle and syringe.

Without intending to be bound by any particular theory, it is believedthat the significant increase in surface area of the resulting bonesubstitute cement as compared to the components thereof, is due, atleast in part, to the reaction of the porogen (e.g., mannitol), acidiccalcium salt (e.g., calcium sulfate dehydrate), and the basic calciumsalt (e.g., calcium carbonate). The bone substitute compositions of thepresent invention have the ability to convert lower surface areastarting materials to high surface area nano-crystallinecalcium-deficient hydroxyapatite (CDHA). The nanostructured nature ofthe resulting CDHA formed due to the reaction and the high specificsurface area, results in faster resorption kinetics of the bonesubstitute cement in-vivo compared to the resorption of in-vivo calciumphosphate cement in published data. The conversion into CDHA may alsoincrease the adsorption amounts of proteins and other growth factors onthe cement.

Furthermore, without intending to be bound by any particular theory, itis believed that the neutral pH, e.g., invariant pH, of the bonesubstitute composition of the present invention may contribute to anincreased rate of bone regeneration. In the prior art, the acidic orbasic bone substitute compositions caused transient cyto-toxicity whichmay adversely affect the adsorption of proteins and growth factors andtherefore, may hinder bone regeneration.

Further, without intending to be bound by any particular theory, it isbelieved that incorporation of nanoparticulate calcium phosphate intothe bone substitute composition is achievable without causing anysignificant change in the physical or chemical characteristics of thecomposition. The nanoparticulate calcium phosphate provides the abilityto bind and condense cellular and biological compounds, such as DNA,growth factors and proteins, in addition to, adding these materials tothe composition. Further, the use of a nanoparticulate calcium phosphatesolution provides the ability to control the delivery of signalingmolecules, anti-biotics, anti-bacterial agents, anti-fungal agents,growth factors and proteins. Moreover, the porosity of the bonesubstitute cement allows the nanoparticate calcium phosphate and thecomplexed cellular and biological material thereto, to be embeddedwithin the open pores formed within the cement. The bone substitutecement includes the presence of macro-pores, micro-pores and meso-pores.The term “macro-pores” refers to pores having a size greater than about15 nm; “micro-pores” refers to pores sized smaller than about 2 nm; and“meso-pores” refers to pores sized in the range of from about 2 to about15 nm in size.

The following examples are merely provided for illustrative purposes andin no way are limiting upon the scope of the present invention.

EXAMPLES Example 1

A powder composition was prepared which included 80% by weight ofα-Ca₃(PO₄)₂, 10% by weight of CaSO₄. 2H₂O and 10% by weight of CaCO₃.Thus, a 500.0 mg sample of the powder composition included 400 mg ofα-Ca₃(PO₄)₂, 50 mg of CaSO₄, 2H₂O and 50 mg of CaCO₃). A solution of0.25M Na₂HPO₄ (pH=9.0) was used as a mixing liquid. The powder to liquidratio was 2.33 gm/cc. The initial (workable time and final (hardening)setting time of the bone substitute composition obtained at 298K usingthe above mentioned powder to ratio was 7 minutes and 18 minutes,respectively. As referred henceforth, this bone substitute compositionis described as CSD-CPH.

Example 2

A powder composition was prepared which included 80% by weight ofα-Ca₃(PO₄)₂, 10% by weight of CaSO₄. 2H₂O and 10% by weight of CaCO₃.Thus, a 500.0 mg sample of the powder composition included 400 mg ofα-Ca₃(PO₄)₂, 50 mg of CaSO₄, 2H₂O and 50 mg of CaCO₃. 50 mg of Na₂HPO₄anhydrous powder was mixed with this 500 mg of powder composition. Acalcium phosphate particulate (CaP) solution based on 0.2M CaCl₂ wasused as a mixing liquid. The powder to liquid ratio was 2.33 gm/cc. Theinitial (workable time) and final (hardening) setting time of the cementobtained at 298K using the above mentioned powder to liquid ratio was 7minutes and 18 minutes, respectively. As referred to henceforth, thisbone substitute composition is described as CSCC-CPH.

Example 3

A powder composition was prepared which included 80% by weight ofα-Ca₃(PO₄)₂, 10% by weight of CaSO₄. 2H₂O and 10% by weight of CaCO₃.Thus, a 300.0 mg sample of the powder composition included 240 mg ofα-Ca₃(PO₄)₂, 30 mg of CaSO₄, 2H₂O and 30 mg of CaCO₃). 50 mg of Na₂HPO₄anhydrous powder. Then, 200 mg of recrystallized mannitol were mixedwith this 300 mg of powder composition. A nano-CaPs solution based on0.2M CaCl₂ was used as a mixing liquid. The powder to liquid ratio was2.33 gm/cc. The initial (workable time) and final (hardening) settingtime of the cement obtained at 298K using the above mentioned powder toliquid ratio was 7 minutes and 18 minutes, respectively. As referral tohenceforth, this bone substitute composition is described as PO-CPH.

Example 4

A powder composition was prepared which included 80% by weight ofα-Ca₃(PO₄)₂, 10% by weight of CaSO₄. 2H₂O and 10% by weight of CaCO₃.Thus, a 300.0 mg sample of the powder composition included 240 mg ofα-Ca₃(PO₄)₂, 30 mg of CaSO₄, 2H₂O and 30 mg of CaCO₃. 50 mg of Na₂HPO₄anhydrous powder. Then, 200 mg of recrystallized mannitol were mixedwith this 300 mg of powder composition. To this 500 mg sample was added15 mg carboxymethyl cellulose (sodium salt) such that the weightpercentage of the carboxymethyl cellulose was 3% based on the totalweight of the 500 mg sample. A nano-CaP solution based on 0.2M CaCl₂ wasused as a mixing liquid. The powder to liquid ratio was 2.10 gm/cc. Theinitial (workable time) and final (hardening) setting time of the cementobtained at 298K using the above mentioned powder to liquid ratio was 9minutes and 50 minutes, respectively. The cohesion time for this cementpaste was 3 minutes. The cement paste obtained by this method wasinjectable and the percentage injectability was greater than 90%. Asreferred to henceforth, this bone substitute composition is described asINJ-PO-CPH.

The crystalline phase evaluations and morphological characteristics ofthe INJ-PO-CPH cement was similar to the PO-CPH and all the resultsdescribed below for PO-CPH are also valid and similar for the INJ-PO-CPHcement.

Compositions including (i) calcium phosphate alone, (ii) calciumphosphate and calcium sulfate, and (iii) calcium phosphate, calciumsulfate and calcium carbonate were prepared and tested to determine thepH of the compositions over time. The results are presented in FIG. 1.As shown in FIG. 1, the addition of calcium sulfate alone (e.g. 80% ofα-Ca₃PO₄)₂ (α-TCP) and 20% of CaSO₄.2H₂O(CSD)) led to a decrease in pHover time, although there was not a significant pH change observedduring the first 60 minutes of the reaction. The bone substitutecompositions of the invention showed no appreciable change in pH over aperiod of two weeks.

The cement in accordance with the present invention CSCC-CPH (seeExample-2) was converted fully into calcium deficient hydroxyapatite(CDHA) as found by measuring powder X-ray diffraction (XRD) and FTIRwithin 15 days of being when kept under Phosphate Buffered Saline (PBS)at 310K.

The surface was area measured using BET by adsorption of nitrogen of theCSCC-CPH powder composition and was found to be approximately 1.0 m²/g.The surface area of the set cement (rod-shaped having of diameter of 6mm and length of 15 mm) was measured following the final setting timewhich was approximately 26 m²/g. The surface area was increased toapproximately 55 m²/g and to 35 m²/g after being kept in PBS for 6 and15 days, respectively, at 310K.

The macro-porosities inside the developed cement PO-CPH (see Example-3)were created using re-crystallized D-Mannitol (porogen) powder. All ofthe mannitol in the Mannitol-cement was leached out within 3 days whenthe pellet was kept under PBS at 310K. This was confirmed by measuringXRD and FTIR. The Mannitol-cement was found to be converted fully intoCDHA as found by XRD after 6 days of being kept under PBS at 310K. The %porosity value obtained by density measurements for the cementcontaining 40% by weight of D-mannitol (Mannitol-cement) was found to be75 (±5%) after the cement pellet was kept inside the phosphate buffersolution (PBS) for 6.0 days at 310K.

The presence of micro and macro-pores within the size range of 10 nm-300μm for the PO-CPH cement after removal of mannitol were confirmed usingMercury-porosiometry and BET. The surface area of the Mannitol-Cementwas measured following the final setting time and was found to beapproximately 11 m²/gm. The surface area had increased to approximately59 m²/g and to 42 m²/g after being kept in PBS for 6 and 15 days,respectively, at 310K. This high surface area for the CSCC-CPH andPO-CPH provided the impetus for the conversion of the micron sizedcement particles into nano-crystalline CDHA. This conversion of themicro size cement particles to nano-size CDHA was further confirmedusing scanning electron microscope (SEM).

Example 5

A cement was developed (not of the invention) based on powders of α-TCPand Na₂HPO₄, comprising α-TCP particles of 7-21 μm in size. Initial andfinal setting time of this cement was found to be 7±1 min and 18±1 min.,respectively, at 298K.

To incorporate micro, meso and macro-porosity into this cement, varyingamounts of CSD were added to the cement. CSD is a relatively solublecalcium rich phase, and thus can be leached out from the cement to formpores. Further, it is contemplated that dissolved CSD in vivo shouldreact with the phosphate ions present in body fluids to form apatitewhich could lead to the replacement of CSD crystals by pores and to thegrowth of neighboring apatite crystals.

The initial and final setting times of these cements (henceforth,described as CSD-CPH) containing varying amounts of CSD were found to bein the range of 9-7 (±1) min and 19-17 (±1) min respectively at 298K.The cohesion time of these cements were found to be ˜4±1 min inphosphate buffer saline (PBS) and the injectability of these cementswere observed to be in the range of 60-70%. CSD was used as the porogenin this example and 80% α-TCP and 20% CSD were used. The details of theprotocol are described above in Example 2.

Detailed studies of the crystalline phase and morphological evolution ofthese CSD-CPH cements with time in PBS were also conducted using X-raydiffraction (XRD) and scanning electron microscopy (SEM). XRD patterns,as shown in FIG. 2, reflect the conversion of the α-TCP phase intocalcium deficient apatite (CDHA) and the dissolution of the CSD withtime. XRD results clearly demonstrate that the α-TCP phase was convertedfully into CDHA after 15.0 days. However, the CSD presented in thecement was completely dissolved in PBS and thus shows no characteristicpeaks after 6.0 days. The SEM images (FIG. 3) show a change inmorphology of the α-TCP particles and preferential growth of CDHAwhiskers on the surface of the α-TCP particles. The cell viability teston the CSD containing cements showed cytotoxic effects of these cementscompared to the CPH cements containing no-CSD. The cytotoxic effects ofthese cements were found to be due to the acidic nature of the cements(found by monitoring the pH changes of the solutions containing cementspowders as shown in FIG. 1).

To avoid transient toxic effects of the acidic CSD-CPH cements in vivo,cements exhibiting neutral pH characteristics were developed. This wasachieved by adding a controlled amount of basic calcium salt, CAC. 10%of CSD and 10% of CAC and 80% of α-TCP (see Example 2) were combinedtogether. The setting characteristics of these CSCC-CPH cements weresimilar to the CSD-CPH cements described above. The phase evaluation ofthe CSCC-CPH cements kept under PBS showed that the complete conversionof the cements into CDHA occurred at approximately 15 days (see FIG. 4(a)). However, the morphology of the as-formed CDHA from these cementswas found to be different from the CSD-CPH cements and the size (lengthand diameter) of the CDHA whiskers formed in case of the CSCC-CPHcements were in the nanometer range. See FIGS. 5( a), 5(b) and 5(c)which show SEM images of the CSCC-CPH cement at aging times in PBS at310K of as-set, after 6 days and after 30 days, respectively. Thisresult suggests that the CSCC-CPH cements may have a better in vivoresorption rate than CSD-CPH cements.

The in vivo resorption rate of cements was further improved by theintroduction of controlled % porosity of micro-pores (to allowcirculation of body fluid) and macro-pores (to provide a scaffold forblood-cell infiltration). It was contemplated that the micro-pores mayfacilitate cell attachment, migration and proliferation enabling gooduptake of the carriers. This was achieved by using water solublere-crystallized Mannitol (porogen). The introduction of 40% (by weight)of the porogen (into the PO-CPH cements) did not alter the initial andfinal setting times, cohesion time, and injectability characteristic ofthese cements (PO-CPH cements, see Example 3). The XRD patterns ofporogen-containing cements showed that they were converted into CDHAwithin 6 days (see FIG. 4( b) which shows PO-CPH cements at varyingaging times in PBS at 310K). The SEM images of the porogen-containingcements showed the formation of micro- and macro-pores after dissolutionof the porogen (see FIGS. 6( a), 6(b), 6(c) and 6(d), which show thePO-CPH cement after aging times, in PBS at 310 K, of as-set, 1 day, 3days and 6 days, respectively).

As used herein, the term “macro-pore” refers to pores having a sizegreater than 15 nm; “micro-pore” refers to pores sized smaller than 2nm; and “meso-pore” refers to pores sized in between the two, i.e.,between about 2-15 nm in size.

The pore size distribution and pore characteristic of the cements, withand without porogens were further studied using mercury-porosimetry,which demonstrated that the 40% Mannitol-containing PO-CPH cementscontained large numbers of macro-pores (between 100 μm and 1 μm)compared to cements with no organic porogens, i.e. CSCC-CPH cements.However, both of these cements contained considerable numbers ofmicro-pores. A description of the surface area, apparent density, andpercentage porosity of these cements are given in Table 1. The surfacearea data (Table1) together with the XRD results demonstrated that allthe cements after 6.0 days of aging in PBS converted intonano-crystalline CDHA. The porosity values of these cements showed thatthe porogen-containing cements were highly porous containing both micro-and macro-pores. It was contemplated that high porosities of theporogen-containing cements together with the formation ofnano-crystalline CDHA may improve the in vivo dissolution rate of thesecements.

TABLE 1 The specific surface area was measured by N₂ adsorption (BET),the apparent density was measured using mercury porosiometry at 30.0 psiand the percentage porosity was measured for the different cementssynthesized. d_(HA) = 3.14 g/cm³ is the crystal density ofhydroxyapatite and d_(CPC) is the apparent density of a dried CPC.Porosity (%) = Apparent density [(d_(HA)-d_(CPC))/d_(HA)] X SamplesSurface area (m²/g) (g/cc) at 24° C. 100 CSCC-CPH-cement- ~25.98 ~1.15~63 0.0 days in PBS CSCC-CPH-cement- ~55.01 ~1.02 ~67 6.0 days in PBSPO-CPH-cements- ~11.56 ~1.12 ~64 0.0 days in PBS PO-CPH-cements- ~59.03~0.72 ~77 6.0 days in PBSComposition of the CSCC-CPH Cements

400 mg of α-Ca₃(PO₄)₂, 50 mg of CaSO₄. 2H₂O and 50 mg of CaCO₃ wasprepared. A 50 mg of Na₂HPO₄ anhydrous powder was mixed with this 500 mgof powder composition. A nano-CaPs solution based on 0.2M CaCl₂ was usedas a mixing liquid. The powder to liquid ratio was 2.33 gm/cc.

Composition of the PO-CPH Cements

240 mg of of α-Ca₃(PO₄)₂, 30 mg of CaSO₄. 2H₂O and 30 mg of CaCO₃ wasprepared. A 50 mg of Na₂HPO₄ anhydrous powder and 200 mg ofrecrystallized mannitol was mixed with this 300 mg of powdercomposition. A nano-CaPs solution based on 0.2M CaCl₂ was used as amixing liquid. The powder to liquid ratio was 2.33 gm/cc.

Injectable Cement Formulation

336 mg of α-Ca₃(PO₄)₂, 42 mg of CaSO₄. 2H₂O and 42 mg of CaCO₃ wasprepared. A 70 mg of Na₂HPO₄ anhydrous powder and 280 mg ofrecrystallized mannitol was mixed with this 420 mg of powdercomposition. Furthermore, 21 mg of carboxymethyl cellulose was added andmixed with the cement-mannitol powder. A nano-CaPs solution based on0.2M CaCl₂ was used as a mixing liquid. The cement powder and nano-CaPssolution were mixed with a powder to liquid ratio of 2.1 gm/cc. Theresults for this injectable formulation were similar to the resultsobtained for the cement formulations in Table 1.

In Vitro Experiments and Results

For all these cements MC3T3-cell proliferation was assessed with thenontoxic Alamar Blue dye and shown in FIG. 7( a). The proliferationresults show that the cells grow well in all these cements, however, theMannitol-cements showed the best cell growth compared to the othercements.

Bovine Serum Albumin (BSA) Release from Cement

The wells of a 96-well dish were coated with an inventive cement, or acommercial cement (Stryker HydroSet™), or contained no cement (tissueculture plastic (TCP)). The three substrates were coated with either ahigh or low concentration of fluorescently labeled bovine serum albumin(BSA). Solutions containing 0.8 or 0.33 mg/ml of BSA in PBS wereprepared. Two hundred and fifty were added to the different substratesresulting in 0.20 and 0.0825 mg BSA/well for high and low BSAconcentrations, respectively, with an n=3. The BSA solutions were lefton the substrates for 2 days at 37° C. The wells were then rinsed for 1minute with release medium containing α-MEM with 10% fetal bovine serum,1% penicillin-streptomyocin, and 1% L-glutamine; the release medium wasthen refreshed. The release medium was removed from each well and thefluorescence was measured using a Perkin Elmer 1420 Victor³V multilabelspectofluorometer after 1 and 4 hours and 1, 4, 7, 13, 21, 28, and 46days. The cumulative BSA released was calculated by taking the sum ofBSA measured from all time points.

FIG. 7( b) shows the release profile of BSA from the cement according tothe present invention (“our cement”), the commercially available cementand the tissue culture plastic (TCP). The commercial cement and TCPadsorbed lower amounts of BSA in comparison to the inventive cement. Thecommercial cement and TCP cement showed an initial release at 1 and 4hours whereas our cement showed a sustained release over a span of 46days.

FIG. 8( a) shows a digital image of the as-prepared cement PO-CPH. Asmentioned above, the cements can be synthesized in non-porous and porousforms. Furthermore, as shown in FIG. 8( b), the cement can be in theform an injectable paste.

3-D Printing of Cement

It was demonstrated to print scaffolds of arbitrary and complex 3Danatomical shapes with hierarchical porous structures mimicking themacroscopic and internal microstructure of organs and tissues whileproviding temporary mechanical function and mass transport propertiesusing the developed cements.

Scaffolds were prepared for in vivo experiments (rather than usinginjectable cement). Scaffolds were printed (see FIG. 9) from CSCC-CPHpowders generated using Example 2. A sample of 4.0 kilogram of CSCC-CPHpowder was used in a 3-D printer (Z-corporation, Z-510) and distilledwater was used as a binder and cement initiator. The printing of thescaffolds were carried out at 310K under laboratory conditions. Theprinted scaffolds were de-powdered and then immersed in PBS for 24 hrsat 310K. The scaffolds were then dried with acetone and sterilized usingethylene oxide for in vivo implantation.

In Vivo Experiments and Results

Methods

Scaffolds Preparation

PO-CPH Scaffolds

240 mg of α-Ca₃(PO₄)₂, 30 mg of CaSO₄. 2H₂O and 30 mg of CaCO₃ weremixed together. 50 mg of Na₂HPO₄ anhydrous powder and 200 mg ofrecrystallized mannitol was mixed with this 300 mg of powdercomposition. A nano-CaPs solution based on 0.2M CaCl₂ was used as amixing liquid. The powder to liquid ratio was 2.2 gm/cc. The resultingcement paste was placed inside a Teflon mold to fabricate rod shapedsamples. The cement paste inside the mold was kept at a temperature of37° C. to harden for 120 minutes. The rod shaped cement scaffold wasremoved from the mold. The diameter and length of these PO-CPH scaffoldswere 6 mm and 15 mm, respectively. The weights of these scaffolds were650±20 mg. These scaffolds were sterilized using gamma-irradiationbefore implantation.

BMP-2 Loaded PO-CPH Scaffolds

The rod shaped PO-CPH scaffolds were prepared and sterilized usinggamma-irradiation. The nano-CaPs loaded with BMP-2 was prepared bymixing 47 μL of BMP-2 (concentration 1.5 mg/mL) with 4.65 μL of 4MCaCl₂. This mixture was then added to 75 μL of 1.91 mM phosphate bufferto form the BMP-2 loaded nano-CaPs. The final volume of theBMP-2-containing nano-CaPs solution was 126.65 μL. All of the liquidsused in this method were sterilized prior to use and were handled understerilized conditions during mixing and handling. This 126.65 μLsolution was carefully soaked into the dry sterilized preformed PO-CPHcement scaffolds and stored at 4° C. The total BMP-2 loading was 70μg/scaffold. In the case of the 3-D printed structures, the sameprocedure was used except that the BMP-2 loading was tested at 70 μg andalso reduced to 35 μg/scaffold.

INJ-PO-CPH paste for Craniofacial Critical Size Defects

336 mg of α-Ca₃(PO₄)₂, 42 mg of CaSO₄. 2H₂O and 42 mg of CaCO₃ wereprepared. 70 mg of Na₂HPO₄ anhydrous powder and 280 mg of recrystallizedmannitol were mixed with this 420 mg of powder composition. Furthermore,21 mg of carboxymethyl cellulose was added and mixed with thecement-mannitol powder. A nano-CaPs solution based on 0.2M CaCl₂ wasused as a mixing liquid. This powder mixture was sterilized usingethylene oxide before implantation. All the liquids were sterilizedusing filtration before the preparation of nano-CaPs. The cement powderand nano-CaPs solution were mixed with a powder to liquid ratio of 2.1gm/cc under sterilized conditions in the surgery room and the paste wasused to fill a craniofacial critical size defect.

BMP-2 Loaded INJ-PO-CPH Paste for Craniofacial Critical Size Defects

336 mg of of α-Ca₃(PO₄)₂, 42 mg of CaSO₄. 2H₂O and 42 mg of CaCO₃ wereprepared. 70 mg of Na₂HPO₄ anhydrous powder and 280 mg of recrystallizedmannitol were mixed with this 420 mg of powder composition. Furthermore,21 mg of carboxymethyl cellulose was added and mixed with thecement-mannitol powder. A nano-CaPs solution based on 0.2M CaCl₂ wasused as a mixing liquid. This powder mixture was sterilized usingethylene oxide before implantation. All the liquids were sterilizedusing filtration before the preparation of nano-CaPs. During nano-CaPspreparation, BMP-2 was added to the CaCl₂ and this solution was addeddropwise to phosphate buffer solution. The cement powder andBMP-2-containing nano-CaPs solution were mixed with a powder to liquidratio of 2.1 gm/cc (Similar to Example-4) under sterilized conditions inthe surgery room and the paste was used to fill a craniofacial criticalsize defect.

Surgical Technique

1. Rabbit Ulna Critical Size Defect

Twelve skeletally mature adult New Zealand White rabbits (˜kg 3.200)were used. Following induction to general anesthesia, a 4 cm lengthanterolateral incision was made over the left forelimb and the tissueoverlying the diaphysis of the ulna was dissected. A critically sizeddefect was created in the ulna of each rabbit forelimb by removing 15 mmof midshaft diaphyseal bone. In the osteotomy space, different types ofscaffolds were implanted: Group 1: CaP cement alone; and Group 2: CaPcement+BMP-2. The soft tissues were approximated with interrupted 4-0Vicryl (Ethicon, Somerville, N.J.) and the skin was closed with 3-0monofilament non-absorbable sutures (Ethilon). All animals received 0.02mg/kg of Ketoprophine (SQ), every 8-12 hours for 3 days postop foranalgesic purposes and postoperative SQ injections (2 mg/kg) of Baytril(Bayer Corp., Shawnee Mission, Kans. 66201) BID for 3 days prophylaxisfor infection. Eight weeks following surgery, all the rabbits wereeuthanatized. The forelimbs were removed, stripped of soft tissues, andprepared for analyses.

2. Craniofacial Critical Size Defect

All rabbits were anesthetized with an IM injection (0.59 ml/kg) of asolution of 91% Ketaset (Ketamine Hydrochloride, 100 mg/ml) and 9%Rompun (Xylazine, 20 mg/ml). The scalps were then shaved, depilated,scrubbed with povidine/alcohol, and prepared for sterile surgery. Thecalvaria were exposed using a midline scalp incision and the skinreflected laterally to the supraorbital borders. Holes were then made inthe periosteum and bone using a fine dental burr (0.4 mm). Then, theholes were connected and the skull was extirpated and removed in onepiece, approximately 15 mm² critical size defect (CSD) was made by usinga dental cutting burr and hand engine. The dura mater was left in situ.

The scaffolds were made under sterile conditions and put into the defectarea. The scalp wounds were closed with 4.0 ethilon sutures. All animalsreceived 0.02 mg/kg of Buprenorphine (SQ), every 8-12 hours for 3 dayspost-op for analgesic purposes. In addition, all animals receivedpostoperative SQ injections (2 mg/kg) of Baytril (Bayer Corp., ShawneeMission, Kans. 66201) BID for 5 days prophylaxis for infection. Skinsutures were removed at 10 days post-operatively.

Body weight, bony defect healing, and cranial vault growth were assessedand monitored at 0, 2, 4, and 6 weeks postoperatively by using serialhead radiographs. At 8 weeks postoperative, all rabbits wereanesthetized with an IM injection of a solution of ketamine (40 mg/kg)and xylazine (7 mg/kg) and euthanized with an IV (300 mg/kg) injectionof pentobarbital (Nembutal, Abbott Laboratories, North Chicago, Ill.).The CSDs were harvested for microCT analysis and histologicalassessment.

3. Subcutaneous Cement Implantation

Adult mice (aged 8-10 weeks) were utilized. The mice were anesthesizedusing Isofluran 2-5%; Ketamine 80-100 mg/kg and Xylazine 2 mg/kg IP werealso used if deemed necessary. The effect of the anesthetic wasdetermined by absent whisker-twitch response and unresponsiveness togentle, passive extremity extension. Ophthalmic ointment was gentlyplaced on the corneas. The surgical site was shaved with an electricclipper, prepped with betadine and draped in standard aseptic fashion.The mice were then numbered using a base three ear notch system.

The operating field was disinfected with 5% solution of iodine. A 1 to 2cm incision on the back of the mice was made and the bone cementimplants were placed into a subcutaneous pocket created in the dorsalarea. The wound was closed with skin staples or sutures (the sutureswere removed on day 7-9). Each animal received 2 or 4 implants.

Imaging Assessment

Animals were followed-up at regular intervals of 2, 4, and 8 weekspostoperatively with radiographs. At 8 weeks computed tomography (CT)using a Scanco Medical AG μCT 40 system followed by histologicalassessment were carried out. The percentage areas of the ulnar criticalsized bone defect occupied by newly formed bone were calculated.

Following euthanasia the explanted forelimbs were scanned. The new boneformation was analyzed by extracting the region of new bone in thecritical size defect using semi-automated contouring.

Non-parametric tests were used and the significant level was consideredat p<0.05.

Histological Assessment and Histomorphometry

Following micro CT scan, the harvested specimens were fixed with 2%paraformaldehyde, decalcified, dehydrated through a graded series ofalcohols and embedded in paraffin using standard tissue processorschedule. Approximately 15 serial sections through the center of thedefect were cut at a thickness of 4-6 micrometers. Histological sectionswere stained by either Hematoxylin and Eosin (H&E) or Masson's Trichromeand examined using a Nikon TE2000 photomicroscope. Quantitativeassessments was achieved using an acquisition, and measurement of newlyformed bone bridge (matrix and cells). These images were analyzed usingthe Bioquant Osteo image analysis software specifically identifyingareas of the tissues, e.g. those mineralized and those not mineralized.A determination of nominal values for histomorphometric assessments wereaccomplished to define the minimal number of fields and sections toassess. A standardized level of magnification were used for analyses anda standard number of microscopic fields and sections were examined foreach experimental cohort at each time. Morphological features within thedefect were identified and enhanced using computerized software in anidentical manner for all experimental replicates. The morphologicalaspects were quantified (bone/mm²) using a Nikon TE2000 photomicroscopefitted with a color digital camera (Qimaging) and integrated with animage analysis system (Bioquant Osteo). Ten percent of the specimenswere scanned and measured twice for inter-rater reliability.

Results

Materials and In-Vitro Characterization

The calcium phosphate cements were fabricated under neutral pHconditions with the direct incorporation of nano-structured calciumphosphates (NanoCaPs) as delivery agents for growth factors and proteinssuch as BMP-2. The cements all showed the formation of nano-structuredhydroxyapatite (HA) as the primary phase after immersion in PBSindicating the probable phase that would likely form in vivo. Thecements also showed excellent cell attachment and cellular migrationresults indicating their strong biocompatibility. The formation ofnano-structured HA and the resulting higher specific surface area wereadded indications of the likely faster resorption kinetics of the cementwhen implanted.

All animals from all the groups wherein scaffolds were implanted,survived until the day of euthanasia. There were no complicationsencountered in terms of local or systemic adverse effects with regard tothe implantation of the scaffolds.

Ulnae Critical Size Detect Results

The results showed that the bone substitute composition prepared inaccordance with the present invention (in the absence of BMP-2) had theability to regenerate an ulnae critical size bone defect. When BMP-2 wasadded to the bone substitute composition, increased bone regenerationwas observed and faster cement resorption was achieved as compared tothe bone substitute composition without the presence of BMP-2. Bothcements with and without BMP-2, yielded higher regeneration potentialthan the control sample of an organic matrix without the BMP-2. Thediscussion below details the radographic, micro-CT and histologicalassessment of the bone regenerative strategy that was employed using themoldable porous calcium phosphate (CaP)-based cements and its results.

Craniofacial Critical Size Defect Results

The results showed that the bone substitute composition prepared inaccordance with the present invention (in the absence of BMP-2) had theability to regenerate a calvarial critical size bone defect. When BMP-2was added to the bone substitute composition, increased boneregeneration was observed and faster cement resorption was achievedcompared to the bone substitute composition without the presence ofBMP-2. Both cements, with and without BMP-2, yielded higher regenerationpotential than the control of organic matrix without BMP-2. Thediscussion below details the radographic, micro-CT and histologicalassessment of the bone regenerative strategy that was employed using themoldable porous CaP-based cements and its results.

Subcutaneous Cement Implantation Results

The data showed that the biocompatible porous cement was infiltrated bycells from the adjacent wound area. These cells, upon their interactionwith the cement, appeared to start differentiating into bone cells asshown by the deposition of an organic matrix that containedextracellular matrix bone proteins such as bone sialoproteins (BSP)(FIG. 14). This data provided support for the osteo-inductivitycharacteristic of the cement to induce cells to differentiate towardsthe bone lineage.

Cement Results

Post implant 8 weeks in vivo results on PCC-CPC without BMP-2 scaffoldsimplanted in an ulnae and craniofacial critical size defect model usingradiographical, micro-CT and histological assessment (FIGS. 10 and 13)demonstrated the formation of new bone on the surface and bulk of theporous scaffold thus indicating rapid dissolution of the cement.

Cement with rhBMP-2 Results

FIG. 11( c) shows the radiographic images of the ulnae immediatelyfollowing post surgery, after 2 weeks and after 8 weeks. The radiographafter 8 weeks shows complete resorption of the cement with the formationof new bone filling the original defect shown in FIG. 11( c). MicroCTanalysis (FIGS. 11 and 13) in both the ulnae and calvarial bones showfull bone regeneration and bridging of the defect with bone as shown byhistological analysis (FIG. 11( a)).

FIG. 12 shows the radiograph of the ulna following 8 weeks of surgicalimplantation of the 3-D printed scaffold structures as described andshown in FIG. 9. The results show the closure of the non-union.

The Subcutaneous cement implantation results demonstrated that thecement is osteo-inductive. FIG. 14 shows an H&E staining of thedecalcified cement implant infiltrated by cells from the adjacent woundarea. The cells have deposited an extracellular matrix that can bevisualized by the H&E stain. This matrix already at 4 weekspost-implantation contains bone specific proteins such as bonesialoproteins (BSP) as shown by immunohistochemistry staining using anani-BSP antibody (FIG. 14).

Whereas particular embodiments of the invention have been describedherein for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details may be made withoutdeparting from the invention as set forth in the appended claims.

What is claimed is:
 1. A method of repairing or replacing defectivenative bone tissue, comprising: preparing a bone substitute composition,comprising: combining calcium phosphate, acidic calcium salt, basiccalcium salt, material selected from the group consisting of monosodiumhydrogen phosphate, disodium hydrogen phosphate and mixtures thereof,and porogen to form a powder component; and mixing the powder componentwith a liquid colloidal mixture comprising nanoparticulate calciumphosphate and calcium salt; obtaining a patient having defective nativebone tissue; and administering in vivo the bone substitute compositionin a form selected from the group consisting of paste, putty and cement,into the patient at the site of the defective native bone tissue.
 2. Themethod of claim 1, wherein the repairing or replacing defective nativebone tissue is carried out in the absence of a biological growthcomponent.
 3. The method of claim 1, wherein the native bone tissue isin an area selected from the group consisting of orthopedic, dental andcraniofacial.
 4. The method of claim 1, wherein the administering of thebone substitute composition is selected from the group consisting ofinjecting and implanting the bone substitute composition into thepatient.
 5. The method of claim 1, wherein surface area of the bonesubstitute composition is greater than surface area of the powdercomponent.
 6. A method for delivering cellular and biological materialto a target site in a patient, comprising: preparing a bone substitutecomposition, comprising: combining calcium phosphate, acidic calciumsalt, basic calcium salt, material selected from the group consisting ofmonosodium hydrogen phosphate, disodium hydrogen phosphate and mixturesthereof, and porogen to form a powder component; and mixing the powdercomponent with a liquid colloidal mixture comprising nanoparticulatecalcium phosphate and calcium salt, the colloidal mixture beingcomplexed with at least one of a compound selected from the groupconsisting of protein, peptide, DNA, drug, stem cell, normal cell, andcombinations thereof; obtaining a patient having defective native bonetissue; and administering in vivo the bone substitute composition in aform selected from the group consisting of paste, putty and cement, intothe patient at the target site.
 7. A bone substitute composition,comprising: a powder component, comprising: calcium phosphate; acidiccalcium salt; basic calcium salt; material selected from the groupconsisting of monosodium hydrogen phosphate, disodium hydrogenphosphate, and mixtures thereof; and porogen; and a liquid component,comprising: a colloidal mixture comprising nanoparticulate calciumphosphate and calcium salt, the nanoparticulate calcium phosphate formedby reaction of calcium and non-acidic ionic phosphate in the presence ofhydroxyl ions, wherein the composition is effective to regenerate bonein the absence of a biological growth component.
 8. The composition ofclaim 7, wherein the calcium phosphate is selected from the groupconsisting of monocalcium monophosphate anhydrite, calciumhydrogenphosphate dihydrate, calcium hydrogenphosphate anhydrite,hydroxyapatite, alpha-tricalcium phosphate, beta-tricalcium phosphate,fluorapatite, octacalcium phosphate , tetracalcium phosphate, carbonatedcalcium phosphate and mixtures thereof.
 9. The composition of claim 8,wherein the acidic calcium salt is selected from the group consisting ofcalcium sulfate, calcium hydrogenphosphate dihydrate, calciumhydrogenphosphate anhydrite, calcium oxalate, calcium citrate, calciumtartrate, calcium picrate and mixtures thereof.
 10. The composition ofclaim 8, wherein the basic calcium salt is selected from the groupconsisting of calcium carbonate, calcium bicarbonate, calciumdihydroxide, and mixtures thereof.
 11. The composition of claim 8,wherein the porogen is selected from the group consisting ofrecrystallized organic salt, recrystallized inorganic salt, engineeredpeptide, natural polymer, a composite of natural and synthetic polymers,synthetic biodegradable polymer, natural extra cellular matrix protein,and mixtures thereof.
 12. The composition of claim 8, wherein thecolloidal mixture comprising nanoparticulate calcium phosphate iscomplexed with at least one of a compound selected from the groupconsisting of protein, peptide, DNA, drug, stem cell, normal cell, andcombinations thereof.
 13. The composition of claim 8, having a porosityof about 80 percent or greater.
 14. The composition of claim 8, whereincalcium ions are present in excess in the reaction as compared tophosphate ions.
 15. A bone substitute composition, comprising: a powdercomponent, comprising: calcium phosphate; acidic calcium salt; basiccalcium salt; material selected from the group consisting of monosodiumhydrogen phosphate, disodium hydrogen phosphate, and mixtures thereof;and porogen; and a liquid component, comprising: a colloidal mixturecomprising nanoparticulate calcium phosphate and calcium salt, thenanoparticulate calcium phosphate formed by reaction of calcium andnon-acidic ionic phosphate in the presence of hydroxyl ions, wherein theporogen is present in an amount such that the composition has a porosityof about 80% or greater.
 16. The composition of claim 15, wherein thecomposition has a surface area from about 60 m²/g to about 120 m²/g.